Nitric oxide (NO) induces vasodilation by activation of soluble guanylyl cyclase (sGC), which in turn produces the second messenger cGMP. Following exposure to NO, sGC becomes desensitized and fails to respond to additional NO stimulation. Until now, the mechanism of sGC desensitization was unknown. We see an interesting parallel between sGC desensitization and clinical development of nitrate tolerance. Prolonged exposure to nitroglycerin (GTN), which induces vasodilation via NO generation, results in the loss of vasodilatory response to NO and nitrates. The mechanism of this nitrate tolerance is not understood, yet it is a major draw back to a widely used cardiovascular therapy. In addition to sGC activation, NO modulates protein function by S-nitrosylation, which is the addition of a NO moiety to the free thiols of cysteine residues. In our quest to understand the mechanism of desensitization of sGC, we discovered recently that sGC is S-nitrosylated in vitro and in intact cells. We further established a cause-effect relationship between S-nitrosylation of sGC and its desensitization, characterized by the loss of NO-stimulation of sGC activity (while the basal activity remains unaltered). Recently, it was shown that GTN treatment increases S-nitrosylation of proteins in tissues. Importantly, we just showed that GTN induces S-nitrosylation and desensitization of sGC in cells. Therefore, our hypothesis is that GTN treatment induces S-nitrosylation of sGC leading to its desensitization, which in turn underlies nitrate tolerance. To explore this provocative hypothesis, we will need to show first that GTN-dependent S-nitrosylation of sGC directly causes desensitization. Second, we will assess whether S-nitrosylation of sGC in vivo (using an S-nitrosylating agent) can mimic the development of nitrate tolerance. Third, we will determine if GTN treatment under conditions known to induce nitrate tolerance S-nitrosylates sGC in vivo thus provoking its failure to respond to NO stimulation. Primary rat aortic smooth muscle cells will be used to characterize the GTN-induced development of S-nitrosylation and desensitization of sGC with biochemical tools. Physiological characterization of the impact of sGC S-nitrosylation on NO-dependent vasodilation will be conducted in vivo with a hamster cheek pouch preparation and later in the rat cremaster muscle. Resistance to nitrate tolerance in vivo will be attempted with sGC mutants of S-nitrosylation, using adenovirus technology. These initial experiments should lay the foundations for a more complete study that will address many more questions: What is the molecular mechanism of sGC desensitization by S-nitrosylation? Is sGC S-nitrosylated under conditions of oxidative stress, and if so, does it contribute to the development of cardiovascular dysfunctions such as atherosclerosis? Could a specific blockade of sGC S-nitrosylation prevent the development of nitrate tolerance or other cardiovascular diseases? This proposal and its future extension could be critical for the development of therapeutic strategies in which sGC and its desensitization are new targets. PUBLIC HEALTH RELEVANCE: Nitroglycerin has been used for more than a century to treat many cardiovascular diseases by relaxing the vasculature. Unfortunately, it induces nitrate tolerance, which means that the organism becomes insensitive to the treatment. We propose to explore the mechanism underlying nitrate tolerance, which until now remains unexplained.